摘要目的 观察异甘草酸镁(magnesium isoglycyrrhizinate,MgIG)对大鼠肝细胞模拟缺血再灌注损伤的细胞凋亡影响及其机制.方法 分离、培养、鉴定大鼠肝细胞,将对数生长期肝细胞,按照数字表法随机分为模拟缺血再灌注损伤模型组和MgIG低浓度组(0.1 g/L)、中浓度组(1g/L)、高浓度组(10 g/L),预处理24 h后,模拟缺氧6h、复氧4h,进行以下实验观察:(1)应用光镜观察细胞的形态改变;(2)应用四甲基偶氮唑盐(MTT)法检测MgIG对肝细胞活力的影响;(3)应用吖啶橙/嗅化乙啶(AO/EB)染色观察细胞的凋亡情况,计算细胞凋亡率;(4)应用RT-qPCR法检测各组bcl-2、bax基因的表达量.结果 (1)MTT法结果表明,一定浓度的MgIG (0.10、1、10 g/L)能提高缺氧-复氧损伤的肝细胞A值(0.285 ±0.017、0.320±0.008、0.341 ±0.019),与模型组比较,差异有统计学意义(P＜0.01);(2)AO/EB染色结果显示,低、中、高浓度组细胞凋亡率(0.5763±0.0405、0.2866±0.0619、0.1261±0.0439)分别低于对照组(0.7124 ±0.0581),差异有统计学意义(P＜0.05);(3)RT-qPCR结果显示低、中、高浓度组bcl-2基因mRNA的表达量(3.350±0.151、9.279±0.350、15.290±0.756)分别高于对照组(0.928±0.068),差异有统计学意义(P＜0.01),低、中、高浓度组Bax mRNA的表达量(0.743±0.035、0.265 ±0.065、0.119 ±0.019)低于对照组(0.945±0.063)差异有统计学意义(P＜0.01).结论 MgIG能提高缺血再灌注损伤的肝细胞活力和降低肝细胞凋亡,作用机制可能与提高bcl-2的mRNA表达、降低bax mRNA的表达有关.

abstractsObjective To observe the effects of magnesium isoglycyrrhizinate (MgIG) on the apoptosis of hepatocytes induced by simulated hypoxia reperfusion in rats and related mechanism.Methods Hepatocytes of the rats were isolated,cultured and identified,then,the liver cells in logarithmic growth phase were randomly divided into 4 groups:the hypoxia reperfusion injury model group,the low MgIG group (0.1 g/L),the moderate MgIG group (1 g/L) and the high MgIG group(10 g/L).Twenty-four hours after pre-treatment (exposure to simulated hypoxia for 6 h and re-perfusion for 4 h),following measurements were performed.① Changes in cell morphology were observed by optical microscopy.② Effects of MgIG on the vitality of hepatocytes were detected by MTT.③Apoptosis and rate of apoptosis were detected and calculated by AO/EB.④The expression levels of bcl-2 and Bax in all the groups were monitored by real time quantitative fluorescence PCR (RT-qPCR).Then,the detected data were analyzed by SPSS17.0.T test was applied for the comparison of the two mean sample lots,and single-factor ANOVA was used for the comparison of multiple samples.Results ①MTT assay indicated that MgIG at some concentrations (0.1,1 and 10 g/L)could improve the A value of the damaged hepatocytes induced by ischemia and re-perfusion(0.285 ± 0.017,0.320 ± 0.008 and 0.341 ± 0.019),as compared with that of the model group,with statistical significance (P ＜ 0.01).②AO/EB staining revealed that apoptosis rates in the low,moderate and high MgIG groups were(0.5763 ± 0.0405,0.2866 ±0.0619 and 0.1261 ±0.0439),which were all lower than that of the control group (0.7124 ± 0.0581),also with statistical significance (P ＜ 0.05).③RT-qPCR monitoring indicated that the expression levels of bcl-2 mRNA in the low,moderate and high MgIG groups (3.350 ± 0.151,9.279 ± 0.350 and 15.290±0.756) were all higher than that of the control group (0.928 ± 0.068),also with statistical significance(P ＜0.01).The expression levels of Bax mRNA in the low,moderate and high MgIG groups (0.743 ± 0.035,0.265 ± 0.065 and 0.119 ± 0.019)were all lower than that of the control group (0.945 ± 0.063),with statistical significance as well (P ＜ 0.01)Conclusions MgIG could increase the vitality of damaged hepatocytes induced by hypoxia-reperfusion and decrease rate of apoptosis,the mechanism of which might be associated with higher expression of bcl-2 mRNA and lower expression of Bax mRNA.